Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function

Abstract

The family of cullin-RING E3 Ligases (CRLs) and the constitutive photomorphogenesis 9 (COP9) signalosome (CSN) form dynamic complexes that mediate ubiquitylation of 20% of the proteome, yet regulation of their assembly/disassembly remains poorly understood. Inositol polyphosphates are highly conserved signaling molecules implicated in diverse cellular processes. We now report that inositol hexakisphosphate (IP6) is a major physiologic determinant of the CRL-CSN interface, which includes a hitherto unidentified electrostatic interaction between the N-terminal acidic tail of CSN subunit 2 (CSN2) and a conserved basic canyon on cullins. IP6, with an EC 50 of 20 nM, acts as an intermolecular "glue," increasing cullin-CSN2 binding affinity by 30-fold, thereby promoting assembly of the inactive CRL-CSN complexes. The IP6 synthase, Ins(1,3,4,5,6)P5 2-kinase (IPPK/IP5K) binds to cullins. Depleting IP5K increases the percentage of neddylated, active Cul1 and Cul4A, and decreases levels of the Cul1/4A substrates p27 and p21. Besides dysregulating CRL-mediated cell proliferation and UV-induced apoptosis, IP5K depletion potentiates by 28-fold the cytotoxic effect of the neddylation inhibitor MLN4924. Thus, IP5K and IP6 are evolutionarily conserved components of the CRL-CSN system and are potential targets for cancer therapy in conjunction with MLN4924.ullin-RING ligases (CRLs), comprising cullins (Cul 1-3, 4A/B, 5, 7, 9), RING finger Roc1/2, and cullin-specific adaptors, form a prominent family of multiprotein E3 ubiquitin ligases that together mediate 20% of proteasomal degradation (1, 2), and are emerging therapeutic targets (3). CRLs require neddylation, the attachment of an ubiquitin-like NEDD8 molecule, for optimal function (4, 5) and are tightly regulated by the constitutive photomorphogenesis 9 (COP9) signalosome (CSN), an eightsubunit deneddylase complex conserved from plants to humans (6, 7). CSN biochemically inhibits but genetically activates . Elucidating the mechanism of binding and dynamic disassembly of the CRL-CSN complexes is therefore key to understand physiological functions of CRLs (6). Despite recent progress in solving the crystal structures of CRLs (1, 15), CSN (16), and the electron microscopy structure of the CSN-CRL1 complex (10), structural elements mediating CRL-CSN interactions remain elusive. Moreover, the molecular switches underlying CRL-CSN complex dynamics are unclear.The inositol polyphosphate pathway interacts with the CRL-CSN complexes via yet unspecified molecular mechanisms. Inositol polyphosphates (IP4, IP5, IP6) are highly conserved signaling molecules generated from the second messenger inositol 1,4,5-trisphosphate (IP3) by a family of inositol phosphate kinases (IPKs), including inositol 1,4,5-triphosphate 3-kinases, inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1), inositol polyphosphate multikinase (IPMK), and inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5K) (17). Majerus and colleagues reported that CSN copurifies with ITPK1 (18), which catalyzes the first committed step in f...

Arabidopsisinositol polyphosphate kinases regulate COP9 signalosome deneddylase functions in phosphate-homeostasis

“…Mammalian IP5K co-precipitates with CSN2/5 whereas IP6K1 interacts with CSN1/2 25,26 . Arabidopsis ITPK1 has been previously reported to associate with CSN1 27 .…”

“…Apparent contradiction between genetic requirements of the CSN-protection versus its deneddylation activity on CRLs forms the basis of the ‘CSN paradox’ 23 . In mammals, locale-specific coordinated activities of IP5K with IP6K1, functional homologs of Arabidopsis IPK1 and ITPK1, respectively provide InsP 6 to strengthen or InsP 7 to loosen CRL-CSN associations 24–26 . Interaction changes affect cellular ratio of neddylated CUL: unneddylated CUL (CUL Nedd8 :CUL) 26 .…”

“…In mammals, locale-specific coordinated activities of IP5K with IP6K1, functional homologs of Arabidopsis IPK1 and ITPK1, respectively provide InsP 6 to strengthen or InsP 7 to loosen CRL-CSN associations 24–26 . Interaction changes affect cellular ratio of neddylated CUL: unneddylated CUL (CUL Nedd8 :CUL) 26 . Here we show that Arabidopsis IPK1-ITPK1 pair retains conserved CSN interactions and modulate CSN5 deneddylase dynamics.…”

CSNAP, the smallest CSN subunit, modulates proteostasis through cullin-RING ubiquitin ligases

“…In line with this assumption, it was demonstrated recently that the presence of unneddylated Cul1 is important for maintaining the substrate receptor pool and promoting rapid assembly and activation of Cul1-Skp1-F-box complexes . Moreover, prolonging CSN-CRL interaction using irreversible neddylation inhibits CRL activity (Scherer, Ding et al, 2016). Likewise, strengthening the CRL-CSN interaction using the metabolite inositol hexakisphosphate promotes CRL inactivation (Scherer et al, 2016).…”

Crystallization and Preliminary X-Ray Diffraction Analysis of a Mammal Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase

“…In concordance with the important functions ascribed to IP 5 2-K in mammals, disruption of its gene produces early lethality and non-viable murine embryos [10]. In addition, IP 5 2-K and IP 6 mediate the assembly of the family of Cullin-RING E3 Ligases (CRL) and the COP9 signalosome (CSN) complexes, which are involved in the ubiquitylation of 20% of the proteome [11]. Apart from the reported functions in mammals, in zebra fish IP 5 2-K has been shown to have a role in the establishment of left-right asymmetry [12].…”